Insulin resistant glucose uptake in vivo is a hallmark of obesity and Type II diabetes. Recent cloning studies reveal that glucose uptake is mediated by a family of glucose transport proteins which are the products of distinct genes. Two glucose transporters identified in insulin responsive tissues are Glut1, also present in non-insulin responsive tissues and Glut4, expressed primarily in muscle and adipose cells. The overall objective of this proposal is to delineate the molecular and cellular mechanisms for insulin resistant glucose uptake in obesity and diabetes. Specific aims are to determine the mechanisms for insulin resistant glucose uptake in skeletal muscle in the high fat-fed rat model of obesity and insulin resistance, and in human obesity and Type II diabetes. The experiments proposed will delineate whether alterations in the expression, subcellular distribution and/or function of specific glucose transporter isoforms contribute to this insulin resistance. Levels of Glut1 and Glut4 polypeptides in skeletal muscle of lean and of high fat-fed obese rats will be measured by immunoblotting. If transporter number is not altered in muscle in spite of insulin resistance in obese rats, alterations in transporter subcellular distribution or function including intrinsic activity, recruitability in response to insulin, and kinetic properties will be studied. Insights into the molecular mechanisms that regulate transporter gene expression will be gleaned by measuring mRNA levels and transcription rates for Glut1 and Glut4. In human studies glucose transporter isoforms in addition to Glut1 and Glut4 which may be important in human skeletal muscle will be identified and localized by immunofluorescence. The relationship between the expression of specific isoforms in muscle and insulin-mediated glucose disposal measured by euglycemic clamp will be determined in lean, obese and Type II diabetic subjects. If in vivo insulin responsiveness does not correspond to levels of expression of transporter isoforms, other mechanisms such as altered insulin-stimulated translocation or activation of transporters will be evaluated. The regulatory effects of body fat distribution, muscle fiber type and therapeutic interventions such as diet, oral hypoglycemic agents, insulin, and exercise on glucose transporter expression, subcellular distribution, and insulin-stimulated recruitment and activation will be investigated. These studies will increase our understanding of the cellular and molecular mechanisms underlying the insulin resistance of obesity and diabetes and the link between obesity and Type II diabetes.